羊痘病毒环介导等温扩增(LAMP)检测方法的建立

摘要

A method for detection of capripoxvirus (CaPV) through the loop-mediated isothermal amplification (LAMP) was established. The conditions for amplification of CaPV DNAs were optimized using LAMP Real Time Turbidimeter LA-320. It was found that at 62 ℃ for 60 min, highly efficient amplification of the goat pox virus(GPV), sheep pox virus(SPV)and bovine lump skin disease virus (LSDV) were achieved. The minimum detection of viral nucleic acid content was up to 3.1× 10-2 pg/μL, 104 times higher than the PCR method recommended by OIE. The method was rapid, specific and easy to operate.%为建立羊痘病毒(CaPV)的环介导等温扩增(LAMP)检测方法,本研究根据GenBank中羊痘病毒的保守基因序列,设计出针对羊痘病毒的LAMP引物,利用LAMP Real Time Turbidimeter LA-320仪优化得到病毒核酸等温扩增最佳条件是62℃恒温反应60 min.在此条件下,病毒核酸的最低检测含量为3.1×10-2 pg/μL,灵敏度比OIE推荐的PCR方法高104倍.添加钙黄绿素(calcein)建立的目测法,还可实现对上述病原体检测结果肉眼观察.本研究获得的CaPV LAMP仪器法和目测法可特异性地扩增羊痘病毒属的山羊痘病毒、绵羊痘病毒及牛疙瘩皮肤病病毒核酸,具有反应快速、特异性强、灵敏度高、操作简便、设备要求低等特点.